Process for purification of neutral human milk oligosaccharide using simulated moving bed chromatography

ABSTRACT

The present application discloses a process for the purification of a neutral human milk oligosaccharide (neutral HMO). The process uses simulated moving bed (SMB) chromatography which allows the continuous purification of large quantities of HMOs with high purity. Contrary to chemical synthesis routes of neutral HMOs, and their subsequent purification, the presented process allows the provision of HMOs free of noxious chemicals, such as e.g. trace amounts of heavy metals or organic solvents. The individual neutral HMO product may be obtained in solid form by spray drying or as a concentrated syrup. The provided neutral HMO is very well-suited for use in food applications.

The present application discloses a process for the purification of aneutral human milk oligosaccharide (neutral HMO). The process usessimulated moving bed (SMB) chromatography which allows the continuouspurification of large quantities of HMOs with high purity. Contrary tochemical synthesis routes of neutral HMOs, and their subsequentpurification, the presented process allows the provision of HMOs free ofnoxious chemicals, such as e.g. trace amounts of heavy metals or organicsolvents. The individual neutral HMO product may be obtained in solidform by spray drying or as a concentrated syrup. The provided neutralHMO is very well-suited for use in food applications.

Human milk represents a complex mixture of carbohydrates, fats,proteins, vitamins, minerals and trace elements. By far the mostpredominant fraction is represented by carbohydrates, which can befurther divided into lactose and more complex oligosaccharides. Whereaslactose is used as an energy source, the complex oligosaccharides arenot metabolized by the infant. The fraction of complex oligosaccharidesaccounts for up to 1/10 of the total carbohydrate fraction and consistsof probably more than 150 different oligosaccharides. The occurrence andconcentration of these complex oligosaccharides are specific to humansand thus cannot be found in large quantities in the milk of othermammals, such as for example domesticated dairy animals.

The existence of these complex oligosaccharides in human milk has beenknown already for a long time and the physiological functions of theseoligosaccharides have been subject to medical research for many decades.For some of the more abundant human milk oligosaccharides, specificfunctions have already been identified.

The limited supply and difficulties of obtaining pure fractions ofindividual HMOs led to the development of chemical routes to some ofthese complex molecules. However, synthesis of HMOs by chemicalsynthesis, enzymatic synthesis or fermentation has proved to bechallenging. At least large-scale quantities as well as qualitiesadequate for food applications have not been able to be provided todate. In this regard, particularly chemical synthetic routes of specificHMOs (e.g. the HMO 2′-fucosyllactose; see WO 2010/115935 A1) involveseveral noxious chemicals, which involve the risk of contamination ofthe final product.

Due to the challenges involved in the chemical synthesis of human milkoligosaccharides, several enzymatic methods and fermentative approacheshave been developed. However, these methods yield complex mixtures ofoligosaccharides i.e. the desired product is contaminated with startingmaterial such as lactose, biosynthetic intermediates and substrates suchas individual monosaccharides and polypeptides etc.

Processes in the state of the art for purifying individualoligosaccharide products from these complex mixtures are technicallycomplex and also uneconomical for food applications. For thepurification of the disaccharides lactose or sucrose from complexmixtures such as whey or molasses, industrial scale processes have beendeveloped which involve multiple crystallizations. The disadvantage ofsaid methods is that they are elaborate and only lead to low yields.

For the purification of complex oligosaccharides, such as certain HMOs,gel-filtration chromatography has been the method of choice until now.The disadvantage of gel-filtration chromatography is that it cannot beefficiently scaled up and it is unsuitable for continuous operation.Thus, gel-filtration chromatography is not economical and renders itimpossible to provide certain HMOs—like the HMO 2′-fucosyllactose—inreasonable amounts and quality to use them in human food.

Simulated moving bed (SMB) chromatography has its roots in thepetrochemical and mineral industries. Today, SMB chromatography is usedby the pharmaceutical industry for the separation of enantiomers fromracemic mixtures. SMB chromatography has already been used for theseparation of the monosaccharide fructose from fructose-glucosesolutions and for the separation of the disaccharide sucrose from sugarbeet or sugar cane syrups on large-scale. However, SMB chromatographyhas not yet been used for the purification of HMOs, or any other complexoligosaccharide, from fermentations yet.

Simulated moving bed (SMB) chromatography was developed as a continuousseparation process analogous to continuous chemical separation processessuch as rectification. In rectification, a countercurrent is establishedbetween the liquid and the gaseous phase, which allows then thecontinuous application of feed and withdrawal of product(s). Inaddition, counter-current chromatographic operations in theory shouldachieve separations superior to conventional crosscurrent operations.However, chromatographic counter-current operations would require themobile and stationary phases to move in opposite directions. Thus, SMBchromatography was developed as a practical solution to the difficultiesrelated to the concept of moving solid chromatography material in acontinuous chromatographic separation process.

The standard SMB concept involves four different zones with fourexternally applied streams: a feed stream comprising the components tobe separated, a desorbent or mobile phase stream, an extract and araffinate stream (with the raffinate stream representing the lessretained component(s)). These liquid streams divide the SMB system intofour different zones (each zone or section can comprise one or morecolumns) with the following tasks: zone I is required for theregeneration of the solid phase, the purpose of zone II is thedesorption of the less strongly desorbed material, the task of zone IIIis the adsorption of the strongly adsorbed material and finally the taskof zone IV is the adsorption of the less adsorptive material. Thus, morestrongly adsorbing components establish a concentration wave in zone IIand are transported to the extract port whereas less strongly adsorbingcomponents migrate towards the raffinate port.

In principle, zone I and IV serve for regeneration of the solid phase(regeneration zones) whereas zones II and III can be regarded as theactual separation zones of the system (separation zones). In addition tothe four liquid streams and resulting zones, the system contains (forthe closed loop operation) a recycling pump for the mobile phase(desorbent), passing the mobile phase through the fixed zones in onedirection. Counter-current flow is then achieved by the periodicalshifting and continuous supply or withdrawal of feed, desorbent andproducts sequentially from one column to the next in the system.

Besides the standard closed loop, 4 zone SMB system, open loop, 3 zonesystems can be used as well. The 3 zone open loop systems are economicalin the case where fresh solvent is rather inexpensive e.g. where wateror water/ethanol is used as mobile phase. By using a 3 zone open loopconfiguration, the regeneration of the liquid phase is no longer needed,thus making zone IV obsolete.

Besides the standard SMB systems for the separation of a two componentmixture also eight-zone closed loop or five zone open loop SMB systemshave been developed for the separation of more than 2 components.

Due to the continuous mode of operation and also the possibility ofusing rather larger column sizes and recycling of the mobile phase, theSMB system can in principle be scaled into production volumes of 100 sof tons.

Starting from this prior art, the technical problem is the provision ofa novel process to provide a neutral HMO in large amounts, with highpurity and free of noxious chemicals.

The technical problem is solved by the process according to claim 1, theneutral HMO according to claim 14 and the use of a neutral HMO accordingto claim 18. The dependent claims display advantageous embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one purification step using simulatedmoving bed chromatography.

FIG. 2 schematically illustrates two subsequent purification steps usingsimulated moving bed chromatography.

FIG. 3 schematically illustrates a preferred purification schemeaccording to the present invention.

FIG. 4 shows the result of an X-ray powder diffraction analysis of twosamples of spray-dried 2′-fucosyllactose according to the presentinvention.

FIG. 5 shows the particle size distribution of spray-dried2′-fucosyllactose according to the present invention (sample #1 andsample #2) determined by laser diffraction.

FIG. 6 shows the biosynthesis of the neutral human milk tetrasaccharidelacto-N-neotetraose 17.

FIG. 7 shows a comparison of two different nanofiltration membranes forthe concentration of a 2′-Fucosyllactose containing solution bynanofiltration.

FIG. 8 shows the HPLC analysis of the feed (FIG. 8A) and raffinate (FIG.8B) of the SMB chromatography of Example 6.

FIG. 9 shows the HPLC analysis of the extract of the SMB chromatographyof Example 6.

FIG. 10 shows the HPLC analysis of the extract of the second SMBchromatography further SMB chromatography according to the invention) ofExample 6,

FIG. 11 shows the HPLC analysis of the feed (FIG. 11A) and raffinate(FIG. 11B) of the SMB chromatography of Example 7.

FIG. 12 shows the HPLC analysis of the extract of the SMB chromatographyof Example 7.

The present invention provides a process for purification of a neutralHMO (e.g. 2′-fucosyllactose) in a continuous chromatography (or in acontinuous manner) from a crude solution comprising the neutral HMO(e.g. 2′-fucosyllactose) and contaminants, wherein the crude solutioncomprising the neutral HMO (e.g. 2′-fucosyllactose) and contaminantscomprises or consists of a solution which is selected from the groupconsisting of microbial fermentation extract, biocatalysis reactionsolution, chemical synthesis solution, and combinations thereof andwherein the purity of the neutral HMO (e.g. 2′-fucosyllactose) in thesolution is <80%. The process is characterized in that the crudesolution is applied to at least one purification step using simulatedmoving bed chromatography. In this way, a purified solution comprisingthe desired neutral HMO (e.g. 2′-fucosyllactose) with a purity of ≥80%is provided.

The process for purification of a neutral HMO in a continuouschromatography may be a process for purification of a neutral HMO in acontinuous manner. In this regard, the neutral HMO may be2′-fucosyllactose or lacto-N-tetraose.

The Applicant has discovered that with the developed process involving apurification step using simulated moving bed chromatography, HMOs may beprovided with high purity, without heavy metal contaminants and in acontinuous manner. Thus, large amounts of high-quality HMOs may beprovided in a very convenient and economical way, e.g. from a crudesolution from microbial fermentation. The inventive process also turnedout to be highly stable even without a step of regeneration of thecolumn material (e.g. cationic column material) used in the simulatedmoving bed chromatography step. In fact, the whole process can beoperated in a stable and continuous manner for several months.

In a preferred embodiment, the purity of the neutral HMO in the crudesolution is ≤70%, ≤60%, ≤50%, ≤40%, ≤30%, ≤20%, ≤10% or ≤5% and/or thepurified solution contains the HMO with a purity of ≥80%, preferably of≥90%. The term “crude solution” refers to a solution containing neutralHMO before the purification step of single moving bed chromatographywhereas the purified solution refers to a solution after the step ofsingle moving bed chromatography.

The at least one simulated moving bed chromatography step may have

-   i) at least 4 columns, preferably at least 8 columns, more    preferably at least 12 columns, wherein at least one column    comprises a weak or strong cation exchange resin, preferably a    cation exchange resin in the H⁺-form or Ca²⁺-form; and/or-   ii) four zones I, II, III and IV with different flow rates; and/or-   iii) an eluent comprising or consisting of water, preferably ethanol    and water, more preferably 5-15 vol.-% ethanol and 85-95 vol.-%    water, most preferably 9-11 vol.-% ethanol and 89-91 vol.-% water,    wherein the eluent optionally further comprising sulphuric acid,    preferably 10 mM sulphuric acid; more preferably 2-5 mM sulphuric    acid; and/or-   iv) an operating temperature of 15° to 60° C., preferably 20° to 55°    C., more preferably 25° to 50° C.

If the HMO to be purified is 2′-fucosyllactose, the at least onesimulated moving bed chromatography step may have

-   i) four zones I, II, III and IV with different flow rates, wherein    the flow rates are preferably: 28-32 ml/min in zone I, 19-23 ml/min    in zone II, 21-25 ml/min in zone III and/or 16-20 ml/min in zone IV;    and/or-   ii) a feed rate of 2-4 ml/min, preferably 3 ml/min; and/or-   iii) an eluent flow rate of 10-13 ml/min, preferably 11.5 ml/min;    and/or-   iv) a switching time of 16-20 min, preferably 17-19 min, more    preferably 18 min.

Preferably, at least one of the columns comprises 0.1 to 5000 kg ofcation exchange resin, preferably 0.2 to 500 kg of cationic exchangeresin, more preferably 0.5 to 50 kg of cation exchange resin, mostpreferably 1.0 to 20 kg of cation exchange resin.

Importantly, scaling-up of the amount of cation exchange material, theflow rate in the different zones, the feed rate, the eluent flow rateand/or the switching time is possible. The scaling-up may be by a factorof 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000or all possible scaling factors in between said values.

In the columns, a strong cation exchange resin may be used as stationaryphase. Preferably, the cation exchange resin is a sulfonic acid resin,more preferably a Purolite® PCR833H (Purolite, Ratingen, Germany),Lewatit MDS 2368 and/or Lewatit MDS 1368 resin. If a cation ion exchangeresin is employed in the columns, it may be regenerated with sulphuricacid. Sulphuric acid can be employed in the eluent, preferably atconcentration of 10 mM sulphuric acid or less. The (strong) cationexchange resin may be present in H⁺-form or in Ca²⁺-form.

Operating temperatures above 60° C. are not preferred during simulatedmoving bed chromatography. It was found that especially in the presenceof a strong cation ion exchange resin (in H⁺-form or Ca²⁺-form) asstationary phase, the applied neutral oligosaccharides weresignificantly destabilized i.e. depolymerized which was detrimental tothe final yield of the neutral HMO.

In an advantageous embodiment of the invention, the invention ischaracterized in that the purified solution is applied to at least onefurther purification step using simulating moving bed chromatography,wherein a purified solution comprising the neutral human milkoligosaccharide with a purity of ≥90%, preferably ≥92%; more preferably≥93% is provided. In particular, the invention yields a HMO product freeof recombinant DNA, and free of host strain proteins.

The further simulated moving bed chromatography may have

-   i) at least 4 columns, preferably at least 8 columns, more    preferably at least 12 columns, wherein at least one column    comprises a weak or strong cation exchange resin, preferably a    cation exchange resin in the H⁺-form or Ca²⁺-form; and/or-   ii) four zones I, II, III and IV with different flow rates, and/or-   iii) an eluent comprising or consisting of water, preferably ethanol    and water, more preferably 5-15 vol.-% ethanol and 85-95 vol.-%    water, most preferably 9-11 vol.-% ethanol and 89-91 vol.-% water,    wherein the eluent optionally further comprises sulphuric acid,    preferably 10 mM sulphuric acid; more preferably 2-5 mM sulphuric    acid, and/or-   iv) an operating temperature of 15° to 60° C., preferably 20° to 55°    C., more preferably 25° to 50° C.

If the HMO to be purified is 2′-fucosyllactose, the further simulatedmoving bed chromatography step may have

-   i) four zones I, II, III and IV with different flow rates, wherein    the flow rates are preferably: 28-32 ml/min in zone I, 19-23 ml/min    in zone II, 21-25 ml/min in zone III and/or 16-20 ml/min in zone IV;    and/or-   ii) a feed rate of 2-4 ml/min, preferably 3 ml/min; and/or-   iii) an eluent flow rate of 10-13 ml/min, preferably 11.5 ml/min;    and/or-   iv) a switching time of 16-20 min, preferably 17-19 min, more    preferably 18 min.

Particularly, at least one of the columns contains 0.1 to 5000 kg ofcation exchange resin, preferably 0.2 to 500 kg of cation exchangeresin, more preferably 0.5 to 50 kg of cation exchange resin, mostpreferably 1.0 to 20 kg of cation exchange resin.

Importantly, scaling-up of the amount of cation exchange material, theflow rate in the different zones, the feed rate, the eluent flow rateand/or the switching time is possible. The scaling-up may be by a factorof 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000or all possible scaling factors in between said values.

After a purification step using simulated moving bed chromatography, thepH of the purified solution may be adjusted to pH 7, preferably byadding a base, more preferably by adding NaOH (e.g. 0.2 M NaOH).

According to the invention, the crude solution containing the neutralHMO and contaminants comprises or consists of a solution which isselected from the group consisting of microbial fermentation, microbialfermentation extract, biocatalysis reaction solution, chemical synthesissolution and combinations thereof. Fermentation as a source of theneutral HMO has the advantage that it is more cost-effective thanchemical synthesis or biocatalysis i.e. enzymatic synthesis. Thus, amicrobial fermentation extract is preferred.

Preferably, before applying the solution to the at least one simulatingmoving bed chromatography step, the solution (preferably a microbialfermentation solution) comprising the neutral HMO is

-   i) filtered or centrifuged to remove the biomass and/or any    insoluble material, preferably filtered with activated carbon,    charcoal, celite and/or by cross-flow filtration to remove any    insoluble material and organic contaminants, more preferably    filtered by cross-flow filtration, most preferably filtered by    cross-flow filtration using a microfiltration membrane; and/or-   ii) applied to at least one purification step using cation and/or    anion exchange chromatography, preferably first at least one cation    exchange chromatography step and then at least one anion exchange    chromatography step.

In a further preferred embodiment, before applying the solution to atleast one purification step using simulated moving bed chromatography orafter a purification step using simulated moving bed chromatography, thesolution containing the neutral HMO is electrodialysed and/ordiafiltered, preferably diafiltered with a nanofiltration membrane, morepreferably diafiltrated with a nanofiltration membrane having a sizeexclusion limit of ≤20 Å. Most preferably, the solution is dialysed to aconductivity of ≤15 mS/cm, preferably ≤10 mS/cm, more preferably ≤5mS/cm.

If the crude solution is dialysed before applying the solution to atleast one purification step using simulated moving bed chromatography,major contaminants depend on the origin of the neutral HMO fractions(i.e. chemical synthesis, biocatalysis or fermentation). Typicalcontaminants are monosaccharides (e.g. glucose, galactose, fucose,etc.), disaccharides (e.g. lactose) and by-products (e.g. lactulose). Incase where fermentation was used as a source of the neutral HMO, thecrude solution usually comprises the employed carbon source (e.g.glycerol, sucrose and/or glucose) as well as by-products of the employedmicrobes (e.g. higher molecular mass oligosaccharides) as contaminants.As further contaminants, also oligosaccharides may be present which aregenerated due to the promiscuity of the used glycosyltransferases (e.g.glycosyltransferases in the synthesizing cell which convert the desiredproduct, the substrate or an intermediate product into a contaminatingoligosaccharide). Said contaminants can efficiently be removed by apurification step using simulated moving bed (SMB) chromatography.

After dialysis, preferably after electrodialysis and/or diafiltration(optionally before applying the solution to the SMB chromatographicprocess), the solution comprising the HMO may be concentrated, inparticular

-   i) to a concentration of ≥50 g/l, ≥100 g/l, preferably ≥200 g/l,    more preferably ≥300 g/l; and/or-   ii) employing a vacuum concentrator; and/or-   iii) by nanofiltration; and/or-   iv) at a temperature of 4° to 50° C., preferably 10° to 45° C.,    optionally 20° to 40° C. or 30° to 35° C.

More preferably, the HMO-comprising fraction is concentrated byemploying nanofiltration. The nanofiltration step can be further used todialyse away contaminating salts. Here, the HMO fraction may firstly beconcentrated by nanofiltration and the resulting concentrated HMOfraction is then subsequently diluted with water, preferablydouble-distilled H₂O (ddH₂O) or deionized water, and then the dilutedHMO fraction may again be concentrated using a nanofiltration membrane.

Concentration by nanofiltration is particularly preferred because anexposure of the neutral HMOs to high temperatures may be dispensed with.Thus, said method of concentration is less destructive to the structureof HMOs than a heat treatment i.e. it does not induce thermal damage tothe neutral HMOs during concentration. An additional advantage ofnanofiltration is that it can be used both for concentrating and fordialysing (diafiltering) the neutral HMOs. In other words, a membraneused in nanofiltration does not have to be exchanged if theconcentration step and the dialysis step are implemented in successionin the inventive process. In addition, the salt concentration of thesolution containing neutral HMOs can be significantly reduced. Thissaves material and time and makes the whole process more economical.Preferably, nanofiltration is combined with electrodialysis. Thiscombination turned out to give excellent results in concentrating anddesalting.

In a preferred embodiment of the invention, the purified solution issterile filtered and/or subjected to endotoxin removal, preferably byfiltration of the purified solution through a 3 kDa filter.

The purified solution may be spray-dried, particularly spray-dried at aconcentration of the neutral HMO of 20-60% (w/v), preferably 30-50%(w/v), more preferably 35-45% (w/v), an inlet temperature of 110-150°C., preferably 120-140° C., more preferably 125-135° C. and/or an outlettemperature of 60-80° C., preferably 65-70° C.

In a preferred embodiment of the inventive process, the neutral HMOwhich is to be purified is a neutral HMO having more than 3monosaccharide units, preferably a neutral human milk trisaccharide,tetrasaccharide, pentasaccharide or hexasaccharide. More preferably, theneutral HMO is selected from the group consisting of 2′-fucosyllactose,3-fucosyllactose, 2′,3-difucosyllactose, lacto-N-triose II,lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I,lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-neofucopentaose V,lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose,3′-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose,para-lacto-N-hexaose, para-lacto-N-neohexaose anddifucosyl-lacto-N-neohexaose. Most preferably, the neutral HMO is2′-fucosyllactose or lacto-N-neotetraose.

Optionally, it is also possible that the neutral HMO is not2′-fucosyllactose.

In an advantageous embodiment of the invention, the inventive process ischaracterized in that the crude solution comprising the neutral humanmilk oligosaccharide and contaminants comprises or consists of amicrobial fermentation extract, wherein the microbial fermentationextract is obtained in at least one step of microbial fermentation whichis preferably followed by at least one step of

-   a) filtration of a solution, preferably the crude solution, to    separate soluble material from insoluble material after the    microbial fermentation; and/or-   b) ion exchange chromatography, preferably cation exchange    chromatography, more preferably cation exchange chromatography    followed by anion exchange chromatography, of a solution, preferably    of a solution obtained in step a); and/or-   c) concentration of a solution, preferably a solution obtained in    step b), more preferably by evaporation of water and/or by    nanofiltration, optionally by concentrating more than once; and/or-   d) dialysis of a solution, preferably of a solution obtained in step    c), more preferably by electrodialysis and/or diafiltration, most    preferably diafiltration with a nanofiltration membrane, optionally    by dialysing more than once; and/or-   e) chromatography of a solution using simulated moving bed    chromatography, preferably a solution obtained in step d); and/or-   f) filtration of a solution, preferably a solution obtained in step    e), to separate neutral human milk oligosaccharide from coloured    contaminants, more preferably by filtration through activated    carbon; and/or-   g) spray-drying a purified solution comprising the neutral human    milk oligosaccharide, preferably a purified solution obtained in    step f).

Most preferably, all steps a) to g) are implemented in succession. Theimplementation of all steps a) to g) in succession has been found to bethe most advantageous embodiment of the inventive process. Said processis cost and time efficient and enables the provision of large quantitiesof highly pure, spray-dried (i.e. amorphous) neutral HMOs from microbialfermentation (extracts). In particular, the concentration and desaltingsteps of the HMO solution using nanofiltration represent extremely costefficient and gentle operating steps preventing undesired by-productformation.

The invention thus provides a neutral HMO which is producible with theinventive process. The neutral HMO (e.g. 2′-fucosyllactose orlacto-N-tetraose) is preferably spray-dried. The purified neutral HMOhas the advantage of being highly pure and being free of heavy metalcontaminants, and/or organic solvents.

The neutral HMO according to the invention can have

-   i) a solid granule form; and/or-   ii) a glass transition temperature of 60 to 90° C., preferably 62 to    88° C., more preferably 64 to 86° C., determined by differential    scanning calorimetry; and/or-   iii) a particle size of 5 to 500 μm, preferably 10 to 300 μm,    determined by laser diffraction; and/or-   iv) a mean particle size of 10 to 100 μm, preferably 20 to 90 μm,    more preferably 30 to 80 μm, most preferably 40 to 70 μm, determined    by laser diffraction; and/or-   v) an amorphous state, preferably an amorphous state with no    characteristic peaks of crystalline matter in X-ray powder    diffraction, and/or-   vi) a moisture content of 10%, preferably 8%, more preferably <5%.

The neutral HMO may be used in medicine, preferably in prophylaxis ortherapy of gastrointestinal disorders. It can also be used in nutrition,preferably medicinal nutrition or dairy nutrition (e.g. cerealproducts).

In a preferred embodiment of the invention, the neutral human milkoligosaccharide is a neutral HMO having more than 3 monosaccharideunits, preferably a neutral human milk trisaccharide, tetrasaccharide,pentasaccharide or hexasaccharide. More preferably, the neutral HMO isselected from the group consisting of 2′-fucosyllactose,3-fucosyllactose, 2′,3-difucosyllactose, lacto-N-triose II,lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I,lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V, lacto-N-neofucopentaose V,lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose,3′-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose,para-lacto-N-hexaose, para-lacto-N-neohexaose anddifucosyl-lacto-N-neohexaose. Most preferably, the neutral HMO is2′-fucosyllactose or lacto-N-neotetraose.

Optionally, it is also possible that the neutral HMO is not2′-fucosyllactose.

Furthermore, it is proposed to use the neutral HMO according to theinvention as additive in food, preferably as additive in human foodand/or pet food, more preferably as additive in human baby food.

With reference to the following Figures and Examples, the subjectaccording to the invention is intended to be explained in more detailwithout wishing to restrict said subject to the special embodimentsshown here.

FIG. 1 schematically illustrates one purification step using simulatedmoving bed chromatography. The simulated moving bed chromatography mayhave e.g. 12 columns in a serial arrangement 1, wherein the arrangementis divided into four different zones I, II, III and IV. The crudesolution containing the neutral HMO 2′-fucosyllactose and contaminantsis applied between zone II and III to the feed entry 2. Extract isremoved from exit 4 between zone I and zone II whereas raffinate isremoved at exit 3 between zone III and zone IV. Raffinate at exit 3contains the purified HMO 2′-fucosyllactose whereas extract at exit 4contains low molecular weight contaminants (e.g. monosaccharides anddisaccharides).

FIG. 2 schematically illustrates two subsequent purification steps usingsimulated moving bed chromatography. Each simulated moving bedchromatography may have e.g. 12 columns in a serial arrangement 1, 1′,wherein each arrangement is divided into four different zones Ia, IIa,IIIa and IVa or zones Ib, IIb, IIIb and IVb, respectively. The crudesolution comprising the neutral HMO 2′-fucosyllactose and contaminantsis applied between zone IIa and IIIa of the first arrangement 1 to thefeed entry 2. Extract is removed at exit 4 between zone Ia and zone IIawhereas raffinate leaves the first serial arrangement 1 at exit 3between zone IIIa and zone IVa and is applied to the second serialarrangement 1′ between zone IIb and IIIb. In the second serialarrangement 1′, extract is removed at exit 6 whereas raffinate isremoved at exit 5 between zone IIIb and zone IVb. Raffinate at exit 5contains highly purified 2′-fucosyllactose whereas extract at exit 6contains high molecular weight contaminants (e.g. higheroligosaccharides).

FIG. 3 schematically illustrates a preferred purification schemeaccording to the present invention. Firstly, a solution 7 containing theneutral HMO 2′-fucosyllactose and contaminants is applied to anelectrodialysis step 8 until a conductivity of ≤0.5 mS/cm is obtained.Said solution is concentrated until the solution has reached aconcentration of 2′-fucosyllactose of approx. 40% (w/v). Subsequently,said solution is applied to at least one purification step usingsimulated moving bed chromatography 9. After the simulated moving bedchromatography, a purified solution comprising 2′-fucosyllactose withhigh purity is obtained. Said pure solution is subjected to sterilefiltration 11 (preferably also endotoxin removal). Before sterilefiltration 11, an additional step of electrodialysis 10 with subsequentconcentration may optionally be performed. After sterile filtration 11,the purified solution comprising 2′-fucosyllactose is subjected to spraydrying 12 and pure, spray dried 2′-fucosyllactose 13 is obtained insolid granule form.

FIG. 4 shows the result of an X-ray powder diffraction analysis of twosamples of spray-dried 2′-fucosyllactose according to the presentinvention (sample #1 and sample #2). The two obtained diffractogramsreveal that both sample #1 and sample #2 are in the fully amorphousstate (no characteristic peaks of crystalline matter).

FIG. 5 shows the particle size distribution of spray-dried2′-fucosyllactose according to the present invention (sample #1 andsample #2) determined by laser diffraction. A mean particle size ofapprox. 68 μm was determined for sample #1. Sample #2 had a meanparticle size of approx. 44 μm. Both values are considered to be highfor a spray-dried product.

FIG. 6 shows the biosynthesis of the neutral human milk tetrasaccharidelacto-N-neotetraose 17. The biosynthesis starts with the disaccharidelactose 14 which is converted by β-1,3-N-acetyl-glucosamintransferase 15to the trisaccharide lacto-N-triose 16. The trisaccharide lacto-N-triose16 is subsequently converted into the tetrasaccharidelacto-N-neotetraose 18 by the enzyme 1,4-galactosyltransferase 17. Invivo, a certain percentage of lacto-N-neotetraose 18 is furtherconverted into larger oligosaccharides 19, 20, 21 by the enzymesβ-1,3-N-acetyl-glucosamintransferase 15 and 1,4-galactosyltransferase17. If the aim of the inventive process is the purification oflacto-N-neotetraose 17, the larger oligonucleotides as well as thesmaller oligonucleotides (educts) lactose 14 and lacto-N-triose 16 maybe present as contaminants 22 in a crude solution containinglacto-N-neotetraose 17. The inventive process enables efficient removalof said contaminants 22.

FIG. 7 shows a comparison of two different nanofiltration membranes forthe concentration of a 2′-Fucosyllactose containing solution bynanofiltration (VCF: volumetric concentration factor, Flux: expressesthe rate at which water permeates the nanofiltration membrane). AlfaLaval nanofiltration membrane type NF99 (NF) and Alfa Lavalnanofiltration membrane type NF99HF were used as nanofiltrationmembrane. It can be seen that at VCF≤6, the NF99HF nanofiltrationmembrane allows a higher flux, i.e. a faster concentration of thesolution.

FIG. 8 shows the HPLC analysis of the feed (FIG. 8A) and raffinate (FIG.8B) of the SMB chromatography of Example 6 (saccharose was used asinternal standard; see FIG. 8A). As analytical column, a ReproSilCarbohydrate column (amino functionalized silica column; 5 μm; 250×4.6mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4ml/min. As eluent, a mixture of acetonitrile and water (68 vol.-%: 32vol.-%) was used. Elution was isocratic. The injection volume was 20 μl.Oven temperature was 35° C. In the SMB chromatography, a strong cationicexchange resin was used which was present in the H⁺-form.

FIG. 9 shows the HPLC analysis of the extract of the SMB chromatographyof Example 6. As analytical column, a ReproSil Carbohydrate column(amino functionalized silica column; 5 μm; 250×4.6 mm; Dr. Maisch GmbH;Ammerbuch) was employed with a flow rate of 1.4 ml/min. As eluent, amixture of acetonitrile and water (68 vol.-%: 32 vol.-%) was used.Elution was isocratic. The injection volume was 20 μl. Oven temperaturewas 35° C. In the SMB chromatography, a strong cationic exchange resinwas used which was present in the H⁺-form.

FIG. 10 shows the HPLC analysis of the extract of the second SMBchromatography (=further SMB chromatography according to the invention)of Example 6. As analytical column, a ReproSil Carbohydrate column(amino functionalized silica column; 5 μm; 250×4.6 mm; Dr. Maisch GmbH;Ammerbuch) was employed with a flow rate of 1.4 ml/min. As eluent, amixture of acetonitrile and water (68 vol.-%: 32 vol.-%) was used.Elution was isocratic. The injection volume was 20 μl. Oven temperaturewas 35° C. In the SMB chromatography, a strong cationic exchange resinwas used which was present in the H⁺-form.

FIG. 11 shows the HPLC analysis of the feed (FIG. 11A) and raffinate(FIG. 11B) of the SMB chromatography of Example 7. As analytical column,a ReproSil Carbohydrate column (amino functionalized silica column; 5μm; 250×4.6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flowrate of 1.4 ml/min. As eluent, a mixture of acetonitrile and water (68vol.-%: 32 vol. %) was used. Elution was isocratic. The injection volumewas 20 μl. Oven temperature was 35° C. In this SMB chromatography, astrong cationic exchange resin was used which was present in theCa²⁺-form.

FIG. 12 shows the HPLC analysis of the extract of the SMB chromatographyof Example 7. As analytical column, a ReproSil Carbohydrate column(amino functionalized silica column; 5 μm; 250×4.6 mm; Dr. Maisch GmbH;Ammerbuch) was employed with a flow rate of 1.4 ml/min. As eluent, amixture of acetonitrile and water (68 vol.-%: 32 vol.-%) was used.Elution was isocratic. The injection volume was 20 μl. Oven temperaturewas 35° C. In this SMB chromatography, a strong cationic exchange resinwas used which was present in the Ca²⁺-form.

EXAMPLE 1 Purification of 2′-Fucosyllactose Using Simulated Moving BedChromatography (SMB Chromatography)

A clear, particle-free solution containing 2′-fucosyllactose at aconcentration of 250 g/L was electrodialysed to 0.5 mS/cm using aPC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany)equipped with PC-Cell E200 membrane stack. Said stack comprised thefollowing membranes: cation exchange membrane CEM: PC SK and the anionexchange membrane AEM:PcAcid60 having a size exclusion limit of 60 Da.

For SMB purification, the 2′-fucosyllactose solution was concentrated to300 g/L employing a vacuum concentrator at 40° C. For the SMBchromatography, a closed loop SMB system equipped with 12 columns(Prosep® columns with the dimensions: 40 mm×740 mm (Latek, Eppelheim,Germany)) arranged in 4 zones was employed. Each column comprised 760 gof Purolite® PCR833H+(Purolite, Ratingen, Germany) strong cation ionexchanger resin.

The system was operated at a temperature of 25° C. with the followingset flow parameters: flow rate zone I was 30.00 ml/min, flow rate zoneII was set to 21.00 ml/min, flow rate zone III was 21.48 ml/min, flowrate of zone IV was set to 18.44 ml/min, feed was set to 3.00 ml/min,eluent flow was set to 11.56 ml/min and switching time was set to 17.92min. As eluent, water with 10% (v/v) food grade ethanol was used.

Major contaminants such as lactose, monosaccharides such as fucose,glucose, galactose and glycerol, were fractioned into the extract.2′-fucosyllactose and larger oligosaccharide contaminants (e.g.difucosyllactose) were fractioned into the raffinate.

The 2′-fucosyllactose was marginally diluted through the SMBpurification step—the concentration of 2′-fucosyllactose in theraffinate was determined at 200 g/l. The pH of the raffinate wasadjusted to pH 7 by using 0.2 N NaOH. At the described settings the SMBsystems could be continuously operated for at least 3 months.

Then, the obtained solution was again subjected to electrodialysis untila conductivity of less than 0.5 mS/cm was obtained and concentrated toobtain a 40% (w/v) 2′-fucosyllactose solution.

The solution was then subjected to sterile filtration and endotoxinremoval by passing the solution through a 3 kDa filter (Pall Microzaultrafiltration hollow fiber module SEP-2013, Pall Corporation,Dreieich).

Using this protocol 2′-fucosyllactose with a purity of 90.4% could beobtained. Major contaminants were 3′-fucosyllactose (2.6%),difucosyllactose (1.5%) and lactose (1.4%). The yield of thepurification was approximately 80%.

EXAMPLE 2 Purification of 2′-Fucosyllactose Using Multicomponent SMBChromatographic Separation

A clear, particle-free solution comprising 2′-fucosyllactose at aconcentration of 250 g/L was electrodialysed to 0.5 mS/cm using aPC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany)equipped with PC-Cell E200 membrane stack. Said stack comprised thefollowing membranes: cation exchange membrane CEM:Pc SK and anionexchange membrane AEM:Pc Acid 60 which possess a size exclusion limit of60 Da.

For SMB purification, the 2′-fucosyllactose solution was concentrated to300 g/L employing a vacuum concentrator at 40° C. For the SMBchromatography, a close loop multicomponent SMB System equipped with 24columns (Prosep® columns with the dimensions: 40 mm×740 mm (Latek,Eppelheim, Germany)) arranged in 2×4 zones was employed. Each columncontained 760 g of Purolite® PCR833H+(Purolite, Ratingen, Germany)strong cation ion exchanger resin.

The system was operated at 25° C. with the following set flowparameters: flow rate zone Ia was 30.00 ml/min, flow rate zone IIa wasset to 21.00 ml/min, flow rate of zone IIIa was set to 21.48 ml/min,flow rate of zone IVa was set to 18.44 ml/min, feed was set to 3.00ml/min, eluent flow was set to 11.56 ml/min and switching time was setto 17.92 min.

The raffinate of the first separation was passed on at a flow rate of3.04 ml/min to a second separation step. The flow rate of zone Ib waskept at 30 ml/min, flow rate of zone IIb was set to 19.61 ml/min, flowrate of zone IIIb was set to 21.63 ml/min, flow rate of zone IVb was setto 18.46 ml/min, eluent flow was set similarly to 11.56 ml/min andswitching time of zones Ib to IVb was 10.46 min.

As eluent water with 10% (v/v) food grade ethanol was used.

In the multicomponent separation, contaminants such as lactose,monosaccharides such as fucose, glucose, galactose and glycerol werefound in the extract of the first separation step and largeroligosaccharide contaminants (e.g. difucosyllactose) were fractionedinto the raffinate of the second separation step.

2′-fucosyllactose was fractioned into the raffinate of the firstseparation step and the extract of the second separation step and wasthus free of low and high molecular weight contaminants.2′-fucosyllactose was only marginally diluted through the SMBpurification step—the concentration of 2′-fucosyllactose in the extractof the second purification step was determined at 200 g/l.

The pH of the raffinate after the first separation step was adjusted topH 7 by using 0.2 N NaOH.

Using this protocol, 2′-fucosyllactose with a purity of 93.0% could beobtained. Major contaminants were 3′-fucosyllactose (1.1%),difucosyllactose (0.9%) and lactose (1.0%).

EXAMPLE 3 Obtaining 2′-Fucosyllactose in Solid Form by Spray Drying

The 2′-fucosyllactose fractions obtained by SMB chromatography wereagain subjected to electrodialysis treatment until a conductivity ofless than 0.5 mS/cm was obtained. The fractions were then concentratedunder vacuum to obtain 2′-fucosyllactose fractions containing 40% (w/v)2′-fucosyllactose. The solutions were subsequently subjected to sterilefiltration and endotoxin removal by passing the solution through a 3 kDafilter (Pall Microza ultrafiltration hollow fiber module SEP-2013, PallCorporation, Dreieich, Germany).

The thus-obtained sterile 2′-fucosyllactose solutions were then spraydried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz,Germany). For spray-drying of the 2′-fucosyllactose, the 40% (w/v)solution was passed under pressure through the spray dryer nozzles withan inlet temperature set to 130° C. The flow was adjusted to maintainingan outlet temperature between 66° C. to 67° C.

Using these settings a spray dried powder with less than 5% moisturecould be obtained. The moisture contents were determined by Karl-Fischertitration.

EXAMPLE 4 Characterisation of the Spray-Dried 2′-Fucosyllactose

1. Differential Scanning Calorimetry (DSC)

A Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used todetermine thermal events of two samples (sample #1 and sample #2) ofspray-dried 2′-fucosyllactose.

Approximately 25 mg of a spray-dried sample was analyzed in crimpedAlcrucibles (Mettler Toledo, Giessen, Germany). The samples were cooledto 0° C. at 10 K/min and reheated to 100° C. at a scanning rate of 10K/min. After cooling down the samples to 0° C. in a second heating cyclethe samples were heated to 150° C. The midpoint of the endothermic shiftof the baseline during the heating scan was taken as Tg. Exothermic andendothermic peaks are reported by means of the peak temperature and thenormalized energy of the event. The results of the DSC analysis of thesamples are summarized in Table 1.

TABLE 1 1^(st) heating scan 2^(nd) heating scan Tg Endotherm exothermTg1 Tg2 exotherm sample ° C. ° C. J/g ° C. J/g ° C. ° C. ° C. J/g #163.9 87.9 −0.5 82.8 0.7 67.4 122.6 n.d. n.d. #2 87.1 n.d. n.d. n.d. n.d.84.6 n.d. 125.9 1.1 n.d.: not detected

DSC analysis of the sample #1 revealed a main glass transition (Tg) at67.4° C. in the 2^(nd) heating scan. A small second Tg was also detectedat 122.6° C. in the 2^(nd) heating scan. The main glass transition wasdetected in the first 1^(st) heating scan followed by an exo- andendothermic event at temperatures above the Tg. These events areattributed to relaxation effects in the sample.

DSC analysis of the sample #2 showed a substantial higher glasstransition (Tg) at 84.6° C. in the 2^(nd) heating scan which was alsodetected in the 1^(st) heating scan. This may point to a lower residualwater content of sample #2 compared to sample #1. Since the glasstransition was detected close to the final temperature of the 1^(st)heating scan, potential relaxation phenomena could not be detected. Inthis sample, a second glass transition could not be detected although asmall exothermic peak at 125.9° C. was visible in the 2^(nd) heatingscan.

2. X-Ray Powder Diffraction

Wide angle X-ray powder diffraction (XRD) was used to study themorphology of the samples #1 and #2. The X-ray diffractometer Empyrean(Panalytical, Almelo, The Netherlands) equipped with a copper anode (45kV, 40 mA, K_(α1) emission at a wavelength of 0.154 nm) and a PIXcel3Ddetector was used. Approximately 100 mg of the spray-dried samples wasanalyzed in reflection mode in the angular range from 5-45° 2θ, with astep size of 0.04° 2θ and a counting time of 100 seconds per step.

XRD analysis of samples #1 and #2 revealed the fully amorphous state ofboth samples and showed no characteristic peaks of crystalline matter(see FIG. 4 for an overlay of both diffractograms).

3. Laser Diffraction

Laser-diffraction measurements were performed using a Partica LA-950Laser Diffraction Particle Size Distribution Analyzer (Horiba, Kyoto,Japan) equipped with a 605 nm laser diode for detecting particles >500nm and 405 nm blue light emitting diode (LED) for detecting particles<500 nm. Isooctane was used as dispersion medium (refractive index of1.391). Since the refractive index of the samples was unknown, therefractive index of sugar (disaccharide) particles was used (1.530). Thesamples were dispersed in isooctane by ultrasonication for up to 5minutes. Prior to measurement, the system was blanked with isooctane.The dispersion of each sample was measured 3 times and the mean valuesand the standard deviation are reported.

The mean particle size (weighted average of particle sizes by volume)and the mode particle size (peak of the distribution) were recorded. Inaddition to the particle distribution by volume (q %), the result wererecorded as:

-   d(v, 10): particle diameter corresponding to 10% of the cumulative    undersize distribution by volume-   d(v, 50): particle diameter corresponding to 50% of the cumulative    undersize distribution by volume-   d(v, 90): particle diameter corresponding to 90% of the cumulative    undersize distribution by volume

The particle size distribution for sample #1 and #2 is shown in FIG. 5.The mode size, which represents the particle size of the highestintensity, is comparable for both samples. Overall, the mean particlesize of 67.85 μm (sample #1) and 43.65 μm (sample #2), respectively, isregarded as unusually high for spray-dried particles. The fractiondetected at higher particle diameters is probably caused by agglomeratedpowder particles.

Table 2 summarizes the particles size characteristics of sample #1 and#2.

TABLE 2 Size sample #1 sample #2 Mean [μm] 67.84 ± 38.12 43.65 ± 0.57Mode [μm] 12.60 ± 0.07  13.92 ± 0.01 D10 [μm] 10.39 ± 0.17  10.65 ± 0.01D50 [μm] 25.67 ± 4.41  19.68 ± 0.03 D90 [μm] 68.13 ± 26.30 52.37 ± 0.76

EXAMPLE 5 Volume Reduction and Desalting of 2′-Fucosyllactose ComprisingSupernatant Using Nanofiltration

For the concentration and desalting of 2′-fucosyllactose containingculture supernatant, an Alfa Laval M-20 membrane filtration moduleequipped with either a NF99 (Alfa Laval NF99) or NF99HF (Alfa LavalNF99HF) nanofiltration membrane was employed. The used 2′-fucosyllactosecontaining culture supernatant (containing 25 g/l 2′-fucosyllactose) wasseparated from the fermentation microbial biomass by using cross-flowfiltration. The molecular cutoff of the cross-flow filtration membranewas 150 kDa (StrassBurger Filter Micro Cross Module® FS10LFC-FUS1582).

The cell free filtrate was then treated with cation ion exchanger(Lewatit S2568 in proton form (Lanxess)) and anion ion exchanger(Lewatit S6368 in carbonate form (Lanxess)) before being subject tonanofiltration. The solution was neutralized after each ion exchangerstep using sodium hydroxide solution or hydrochloric acid, respectively.Inlet pressure of the membrane module was 42 bar and outlet pressure was40 bar, flow rate of the feed within the membrane module was 8liter/min. The permeate was removed from the process, whereas theretendate was fed back into reservoir and membrane module. The volume ofthe reservoir connected to the membrane stack was 6 liters. During theconcentration of the 2′-fucosyllatose-comprising solution the reservoirwas continuously filled with 2′-fucosyllactose culture supernatantsolution until an 8-fold concentrated solution was obtained.

FIG. 7 shows the obtained flux (L/m²/h) plotted against the volumeconcentration factor (VCF) of the two employed membranes. Havingconcentrated the 2′-fucosyllactose concentration 8-fold to approximately200 g/l 2′-fucosyllactose, the concentrated solution was diafiltered fordesalting by adding deionized water at the same rate as permeate wasobtained from the membrane module. Using the diafiltration step, theconductivity of the 2′-fucosyllactose concentrate could decrease from 25mS to less than 7 mS using the HF99HF membrane.

By using this nanofiltration approach, the 2′-fucosyllactose solutioncould be concentrated 8-fold to a 2′-fucosyllactose concentration of 200g/l 2′-fucosyllactose under mild conditions (avoiding thermal exposure),with the diafiltration step a large fraction of the salt content couldbe removed. The 2′-fucosyllactose concentrate was subjected toelectrodialysis for further reduction of the salt content.

EXAMPLE 6 Purification of Lacto-N-Tetraose Using Two Simulated MovingBed Chromatography Steps and an Ion Exchanger Resin in H⁺ Form

A clear particle free lacto-N-tetraose solution (30 g/l) obtained frombacterial fermentation was electrodialysed to a conductivity of 0.5mS/cm using a PC-Cell electrodialysis apparatus (see above). For SMBchromatography, the lacto-N-tetraose solution was concentrated to 50 g/lunder vacuum at 40° C. Alternatively, the lacto-N-tetraose containingsolution can be desalted and concentrated using a nanofiltrationmembrane (e.g. nanofiltration membrane HF99HF from Alfa Laval).

For SMB chromatography, a closed loop SMB system equipped with 12columns (Prosep® glas columns with the dimensions: 40 mm×740 mm (Lartek,Eppelheim, Germany) arranged in 4 zones was employed. Each glass columncontained 760 g of Purolite® PCR833H+ strong cation ion exchanger resin.The strong cationic exchanger resin was present in the H⁺-form.

The system was operated at 25° C. with the following parameter settings:flow rate of zone I was set to 30.00 ml/min, flow rate of zone II wasset to 19.07 ml/min, the flow rate of zone IV was set to 18.44 ml/min.Feed was kept at 1 ml/min and eluent flow was set to 11.56 ml/min with aswitching time of 17.92 min. As eluent water with 10% (v/v) food gradeethanol was used.

Under these parameters, the lacto-N-tetraose and larger neutraloligosaccharides were fractioned into the raffinate. The purity oflacto-N-tetraose was 86.3% instead of 33.4% at the SMB feed (see FIG. 7Afor HPLC analysis of the SMB feed and FIG. 7B for HPLC analysis of theSMB raffinate). Contaminants such as lactose, monosaccharides andglycerol were found in the SMB extract fraction (see FIG. 8 for HPLCanalysis of the SMB extract fraction). The raffinate containing thelacto-N-tetraose was adjusted to neutral pH using a 0.2 N NaOH solution.

The pH of the obtained extract containing the lacto-N-tetraose wasadjusted to pH 7 by using 0.2 N NaOH. Then, the obtained solution wassubjected to electrodialysis until a conductivity of less than 0.5 mSwas again obtained. The solution was then concentrated under vacuum toapprox. 50 g/l lacto-N-tetraose and then sterile-filtered by passingthrough a 3 kDa cross-flow filter (Pall Microza ultrafiltration hollowfiber module SEP-2013, Pall Corporation, Dreieich).

In order to separate away larger oligosaccharide contaminants from thelacto-N-tetraose, a second SMB chromatography separation was performed.Using the same SMB system the parameters were changed as follows: flowrate zone I 30 ml/min, flow rate zone II 19.27 ml/min, flow rate zone IV17.30 ml/min. Feed was set to 2.08 ml/min and eluent flow 12.70 ml/min.Raffinate flow was 4.04 ml/min and extract flow was 10.73 ml/min.Switching time of the SMB separation was set to 10.46 min. As eluentagain a water/ethanol mixture 9:1 (v/v) was employed. The HPLC analysisof the extract of the second lacto-N-tetraose separation by SMBchromatography is shown in FIG. 10.

Under these conditions, the lacto-N-tetraose was fractioned into theextract and 5 to 10% of the total amount of larger neutraloligosaccharides was found in the raffinate. Using this protocol (aminofunctionalized silica column after second SMB step), lacto-N-tetraosewith a purity of 93.1% could be obtained.

EXAMPLE 7 Purification of Lacto-N-Tetraose Using Simulated Moving BedChromatography with an Ion Exchanger Resin in Ca²⁺ Form

A clear particle free lacto-N-tetraose solution (30 g/l) obtained frombacterial fermentation was electrodialysed to a conductivity of 0.5mS/cm using a PC-Cell electrodialysis apparatus (see above).

For SMB chromatography with calcium as counter-ion, the lacto-N-tetraosesolution was concentrated to 50 g/l under vacuum at 40° C. For SMBchromatography a close loop SMB system equipped with 12 columns (Prosep®glas columns with the dimensions: 40 mm×740 mm (Lartek, Eppelheim,Germany)) arranged in 4 zones was employed.

Each column contained 760 g of Purolite® PCR833H+ strong cationic ionexchanger resin. The cationic ion exchanger resin was washed with 50Liter of a 200 mM CaCl₂ to exchange the H⁺ ions by Ca²⁺ ions. Thus, thestrong cationic exchanger resin was present in the Ca²⁺-form.

The system was operated at 25° C. with the following parameter settings:flow rate zone I was set to 30.00 ml/min, flow rate of zone II was setto 20.07 ml/min, the flow rate of zone IV was set to 17.04 ml/min. Feedwas kept at 2.5 ml/min and eluent flow was set to 11.56 ml/min with aswitching time of 17.92 min. As eluent water with 10% (v/v) food gradeethanol was used.

EXAMPLE 8 Obtaining Lacto-N-Tetraose in Solid Form by Spray Drying

Fractions containing lacto-N-tetraose were concentrated under vacuum toobtain a lacto-N-tetraose concentration of 25% (w/v). The solutions werethen for sterile filtration and endotoxin removal passed through a 3 kDafilter (Pall Microza ultrafiltration hollow fiber module SEP-1013, PallCorporation, Dreieich, Germany).

The thus-obtained sterile lacto-N-tetraose solution was then spray driedusing a Mini Spray Dryer B-290 (Buchi Labortechnik GmbH, Essen,Germany). For the spray-drying of the lacto-N-tetraose, the solution waspassed under pressure through the spray dryer nozzles with an outlettemperature between 120° C. and 130° C. and flow was adjusted tomaintaining an exhaust temperature between 66° C. to 67° C. Using thesesettings, a spray-dried powder with 7-9% moisture and a yield of 72%spray dried lacto-N-tetraose could be obtained. The moisture contentswere determined by Karl-Fischer titration.

The invention claimed is:
 1. A process for purification of a neutralhuman milk oligosaccharide from a crude solution, comprising i.obtaining a crude solution from microbial fermentation, chemicalsynthesis, enzymatic biocatalysis and/or combinations thereof, whereinthe crude solution comprises the-neutral human milk oligosaccharide andcontaminants, and wherein the contaminants in the crude solutioncomprise oligosaccharides generated by a glycosyltransferase used in thesynthesis of the neutral human milk oligosaccharide, ii. applying thecrude solution to simulated moving bed chromatography, wherein eachcolumn in the simulated moving bed chromatography comprises a cationexchange resin, and iii. obtaining a purified solution comprising theneutral human milk oligosaccharide in a raffinate stream, wherein theneutral human milk oligosaccharide has more than three monosaccharideunits.
 2. The process of claim 1, the neutral human milk oligosaccharidein the purified solution has a purity of ≥80%.
 3. The process of claim1, wherein the eluent comprises water and wherein the simulated movingbed chromatography is performed at an operating temperature of 15° to60° C.
 4. The process of claim 1, wherein the simulated moving bedchromatography is operated continuously.
 5. The process of claim 1,wherein the crude solution is obtained by microbial fermentation by amethod comprising a) filtering the microbial fermentation solution toseparate soluble material from insoluble material; b) subjecting thefiltered solution from step a) to at least one of cation exchangechromatography or anion exchange chromatography; c) concentrating thesolution obtained in step b); and d) dialysing the solution obtained instep c) to obtain the crude solution.
 6. The process of claim 1, whereinbefore applying the crude solution to the simulated moving bedchromatography system, the crude solution is i) filtered to remove anyinsoluble material, and/or ii) subjected to at least one of cation oranion exchange chromatography.
 7. The process of claim 1, wherein beforeapplying the crude solution to the simulated moving bed chromatographysystem, the crude solution is i) electrodialysed and/or ii) diafiltered.8. The process of claim 1, wherein before applying the crude solution tothe simulated moving bed chromatography system, the crude solution isdialysed and then concentrated to a concentration of ≥100 g/L.
 9. Theprocess of claim 1, further comprising sterile filtering and/orsubjecting the purified solution to endotoxin removal.
 10. The processof claim 1, wherein the purified solution is concentrated to obtain aconcentrated solution comprising 20-60% (w/v) of the purified neutralhuman milk oligosaccharide, and wherein the concentrated solution isfurther spray dried to obtain the purified neutral human milkoligosaccharide in solid granule or power form.
 11. The process of claim1, wherein the neutral human milk oligosaccharide is selected from thegroup consisting of 2′3-difucosyllactose, lacto-N-tetraose,lacto-N-neotetraose, lacto-N-fucopentaose, lacto-N-neofucopentaose I,lacto-N fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaoseV, lacto-N-neofucopentaose V, lacto-N-difucohexaose I,lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose,lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose,para-lacto-N-neohexaose, and difocosyl-lacto-N-neohexaose.
 12. Theprocess of claim 11, wherein the neutral human milk oligosaccharide islacto-N-tetraose.
 13. The process of claim 8, wherein the crude solutionis dialysed to a conductivity of ≤15 mS/cm.
 14. The process of claim 1,wherein the purified solution is further i) dialysed and/or ii)diafiltered.
 15. The process of claim 14, wherein the purified solutionis electrodialysed to a conductivity of ≤15 mS/cm.
 16. The process ofclaim 3, wherein the eluent comprises water and ethanol.
 17. The processof claim 3, wherein the operating temperature of the simulated movingbed chromatography is 25° to 50° C.
 18. The process of claim 1, whereinthe simulated moving bed chromatography system comprises four zones,wherein each zone has at least two columns.
 19. The process of claim 18,wherein the columns in each of the four zones have flow rates asfollows: a. 28-32 ml/min in zone I; b. 19-23 ml/min in zone II; c. 21-25ml/min in zone III; and d. 16-20 ml/min in zone IV.
 20. The process ofclaim 19, wherein the feed rate of the crude solution is 2-4 ml/min andflow rate of the eluent is 10-13 ml/min.
 21. The process of claim 19,wherein all the columns in the four zones comprise a cation exchangeresin and wherein the raffinate is removed between zone III and zone IV.22. The process of claim 1, wherein the simulated moving bedchromatography system has eight zones.
 23. The process of claim 1,wherein the cation exchange resin comprises hydrogen or calcium cations.24. The process of claim 1, wherein the cation exchange resin comprisessulfonic acid groups.
 25. The process of claim 1, wherein the neutralhuman milk oligosaccharide is a tetrasaccharide, pentasaccharide, orhexasaccharide.
 26. The process of claim 21, wherein the cation exchangeresin comprises hydrogen or calcium cations.
 27. The process of claim21, wherein the cation exchange resin comprises sulfonic acid groups.28. The process of claim 5, wherein all the columns in the simulatedmoving bed chromatography comprises a cation exchange resin.